A novel romaine lettuce cultivar, designated Green Thunder, is disclosed. The invention relates to the seeds of lettuce cultivar Green Thunder, to the plants of lettuce cultivar Green Thunder and to methods for producing a lettuce plant by crossing the cultivar Green Thunder with itself or another lettuce...http://www.google.com/patents/US7348472?utm_source=gb-gplus-sharePatent US7348472 - Lettuce cultivar green thunder

A novel romaine lettuce cultivar, designated Green Thunder, is disclosed. The invention relates to the seeds of lettuce cultivar Green Thunder, to the plants of lettuce cultivar Green Thunder and to methods for producing a lettuce plant by crossing the cultivar Green Thunder with itself or another lettuce cultivar. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other lettuce cultivars derived from the cultivar Green Thunder.

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Claims(26)

1. A seed of lettuce cultivar Green Thunder, wherein a representative sample of seed of said cultivar was deposited under ATCC Accession No. PTA-8605.

2. A lettuce plant, or a part thereof, produced by growing the seed of claim 1.

3. A tissue culture of cells produced from the plant of claim 2, wherein said cells of the tissue culture are produced from a plant part selected from the group consisting of leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, anther, pistil, flower, and stem.

4. A protoplast produced from the plant of claim 2.

5. A protoplast produced from the tissue culture of claim 3.

6. A lettuce plant regenerated from the tissue culture of claim 3, wherein the plant has all the morphological and physiological characteristics of cultivar Green Thunder.

7. A method for producing an F1 hybrid lettuce seed, wherein the method comprises crossing the plant of claim 2 with a different lettuce plant and harvesting the resultant F1 hybrid lettuce seed.

8. A hybrid lettuce seed produced by the method of claim 7.

9. A hybrid lettuce plant, or a part thereof, produced by growing said hybrid seed of claim 8.

10. A method for producing a male sterile lettuce plant wherein the method comprises transforming the lettuce plant of claim 2 with a nucleic acid molecule.

11. A male sterile lettuce plant produced by the method of claim 10.

12. A method for producing an herbicide resistant lettuce plant wherein the method comprises transforming the lettuce plant of claim 2 with a transgene wherein the transgene confers resistance to an herbicide selected from the group consisting of imidazolinone, sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.

13. An herbicide resistant lettuce plant produced by the method of claim 12.

14. A method of producing an insect resistant lettuce plant wherein the method comprises transforming the lettuce plant of claim 2 with a transgene that confers insect resistance.

15. An insect resistance lettuce plant produced by the method of claim 14.

17. A method of producing a disease resistant lettuce plant wherein the method comprises transforming the lettuce plant of claim 2 with a transgene that confers disease resistance.

18. A disease resistant lettuce plant produced by the method of claim 17.

19. A method of producing a lettuce plant with a value-added trait, wherein the method comprises transforming the lettuce plant of claim 2 with a transgene encoding a protein selected from the group consisting of a ferritin, a nitrate reductase and a monellin.

20. A lettuce plant with a value-added trait produced by the method of claim 19.

21. A lettuce plant, or a part thereof, having all the physiological and morphological characteristics of the cultivar Green Thunder, wherein a representative sample of seed of said cultivar was deposited under ATCC Accession No. PTA-8605.

a. crossing a Green Thunder plant, wherein a representative sample of seed was deposited under ATCC Accession No. PTA-8605, with a plant of another lettuce cultivar that comprises a desired trait to produce progeny plants, wherein the desired trait is selected from the group consisting of male sterility, herbicide resistance, insect resistance, and resistance to bacterial disease, fungal disease, or viral disease;

d. selecting for backcross progeny plants that have the desired trait and physiological and morphological characteristics of lettuce cultivar Green Thunder listed in Table 1; and

e. repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny plants that comprise the desired trait and all of the physiological and morphological characteristics of lettuce cultivar Green Thunder listed in Table 1.

23. A plant produced by the method of claim 22, wherein the plant has the desired trait and all of the physiological and morphological characteristics of lettuce cultivar Green Thunder listed in Table 1.

24. The plant of claim 23, wherein the desired trait is herbicide resistance and the resistance is conferred to an herbicide selected from the group consisting of imidazolinone, sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.

25. The plant of claim 23, wherein the desired trait is insect resistance and the insect resistance is conferred by a transgene encoding a Bacillus thuringiensis endotoxin.

26. The plant of claim 23, wherein the desired trait is male sterility and the trait is conferred by a nucleic acid molecule.

Description

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive Romaine lettuce (Lactuca sativa) cultivar, designated Green Thunder. All publications cited in this application are herein incorporated by reference.

There are numerous steps in the development of any novel, desirable plant germplasm. Plant breeding begins with the analysis and definition of problems and weaknesses of the current germplasm, the establishment of program goals, and the definition of specific breeding objectives. The next step is selection of germplasm that possess the traits to meet the program goals. The goal is to combine in a single variety or hybrid an improved combination of desirable traits from the parental germplasm. These important traits may include increased head size and weight, higher seed yield, improved color, resistance to diseases and insects, tolerance to drought and heat, and better agronomic quality.

Practically speaking, all cultivated forms of lettuce belong to the highly polymorphic species Lactuca sativa that is grown for its edible head and leaves. As a crop, lettuces are grown commercially wherever environmental conditions permit the production of an economically viable yield. Lettuce is the world's most popular salad. In the United States, the principal growing regions are California and Arizona which produce approximately 287,000 acres out of a total annual acreage of more than 300,000 acres (USDA, 2001). Fresh lettuces are available in the United States year-round although the greatest supply is from May through October. For planting purposes, the lettuce season is typically divided into three categories, early, mid and late, with the coastal areas planting from January to August, and the desert regions from August to December. Fresh lettuces are consumed nearly exclusively as fresh, raw product, occasionally as a cooked vegetable.

Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositae family). Lettuce is related to chicory, sunflower, aster, dandelion, artichoke and chrysanthemum. Sativa is one of about 300 species in the genus Lactuca. There are seven different morphological types of lettuces. The Crisphead group includes the iceberg and batavian types. Iceberg lettuce has a large, firm head with a crisp texture and a white or creamy yellow interior. Batavian lettuce predates the iceberg type and has a smaller and less firm head. The Butterhead group has a small, soft head with an almost oily texture. Romaine lettuce, also known as cos lettuce, has elongated upright leaves forming a loose, loaf shaped head. The outer leaves are usually dark green. The Leaf lettuces come in many varieties, none of which form a head. The next three types are seldom seen in the United States: Latin lettuce looks like a cross between romaine and butterhead; stem lettuce has long, narrow leaves and thick, edible stems, and Oilseed lettuce is a type grown for its large seeds that are pressed to obtain oil.

Lactuca sativa is a simple diploid species with nine pairs of chromosomes. Lettuce is an obligate self-pollinating species. This means that the pollen is shed before stigma emergence, assuring 100% self-fertilization. Since each lettuce flower is an aggregate of about 10-20 individual florets (typical of the Compositae family), manual removal of the anther tubes containing the pollen is tedious. As such, a modified method of misting to wash the pollen off prior to fertilization is needed to assure crossing or hybridization. About 60-90 min past sunrise, flowers to be used for crossings are selected. The basis for selection are open flowers, with the stigma emerged and the pollen visibly attached to the single stigma (about 10-20 stigma). Using 3-4 pumps of water from a regular spray bottle, the pollen grains are washed off with enough pressure to dislodge the pollen grains, but not enough to damage the style. Excess water is dried off with clean paper towels. About 30 min later the styles should spring back up and the two lobes of the stigma are visibly open in a “V” shape. Pollen from another variety or donor parent is then introduced by gently rubbing the stigma and style of the donor parent to the maternal parent. Tags with the pertinent information on date and pedigree are then secured to the flowers

Choice of breeding or selection methods depends on the mode of plant reproduction, the heritability of the trait(s) being improved, and the type of cultivar used commercially (e.g., F1 hybrid cultivar, pureline cultivar, etc.). For highly heritable traits, a choice of superior individual plants evaluated at a single location will be effective, whereas for traits with low heritability, selection should be based on mean values obtained from replicated evaluations of families of related plants. Popular selection methods commonly include pedigree selection, modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of breeding method. Backcross breeding is used to transfer one or a few favorable genes for a highly heritable trait into a desirable cultivar. This approach has been used extensively for breeding disease-resistant cultivars. Various recurrent selection techniques are used to improve quantitatively inherited traits controlled by numerous genes. The use of recurrent selection in self-pollinating crops depends on the ease of pollination, the frequency of successful hybrids from each pollination, and the number of hybrid offspring from each successful cross

Each breeding program should include a periodic, objective evaluation of the efficiency of the breeding procedure. Evaluation criteria vary depending on the goal and objectives, but should include gain from selection per year based on comparisons to an appropriate standard, overall value of the advanced breeding lines, and number of successful cultivars produced per unit of input (e.g., per year, per dollar expended, etc.). Promising advanced breeding lines are thoroughly tested and compared to appropriate standards in environments representative of the commercial target area(s) for three years at least. The best lines are candidates for new commercial cultivars; those still deficient in a few traits are used as parents to produce new populations for further selection. These processes, which lead to the final step of marketing and distribution, usually take from eight to 12 years from the time the first cross is made. Therefore, development of new cultivars is a time-consuming process that requires precise forward planning, efficient use of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that are genetically superior, because for most traits the true genotypic value is masked by other confounding plant traits or environmental factors. One method of identifying a superior plant is to observe its performance relative to other experimental plants and to a widely grown standard cultivar. If a single observation is inconclusive, replicated observations provide a better estimate of its genetic worth.

The goal of lettuce plant breeding is to develop new, unique and superior lettuce cultivars. The breeder initially selects and crosses two or more parental cultivars, followed by repeated selfing and selection, producing many new genetic combinations. The breeder can theoretically generate billions of different genetic combinations via crossing, selfing and mutations. The breeder has no direct control at the cellular level. Therefore, two breeders will never develop the same cultivar, or even very similar cultivars, having the same lettuce traits.

Each year, the plant breeder selects the germplasm to advance to the next generation. This germplasm is grown under unique and different geographical, climatic and soil conditions and further selections are then made, during and at the end of the growing season. The cultivars that are developed are unpredictable because the breeder's selection occurs in unique environments, with no control at the DNA level (using conventional breeding procedures), and with millions of different possible genetic combinations being generated. A breeder of ordinary skill in the art cannot predict the final resulting cultivars he develops, except possibly in a very gross and general fashion. The same breeder cannot produce the same cultivar twice by using the exact same original parents and the same selection techniques. This unpredictability results in the expenditure of large research monies to develop superior lettuce cultivars.

The development of commercial lettuce cultivars requires the development of lettuce varieties, the crossing of these varieties, and the evaluation of the crosses. Pedigree breeding and recurrent selection breeding methods are used to develop cultivars from breeding populations. Breeding programs combine desirable traits from two or more varieties or various broad-based sources into breeding pools from which cultivars are developed by selfing and selection of desired phenotypes. The new cultivars are crossed with other varieties and the hybrids from these crosses are evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement of self-pollinating crops or inbred cultivars of cross-pollinating crops. Two parents which possess favorable, complementary traits are crossed to produce an F1. An F2 population is produced by selfing one or several F1s or by intercrossing two F1s (sib mating). Selection of the best individuals is usually begun in the F2 population; then, beginning in the F3, the best individuals in the best families are selected. Replicated testing of families, or hybrid combinations involving individuals of these families, often follows in the F4 generation to improve the effectiveness of selection for traits with low heritability. At an advanced stage of inbreeding (i.e., F6 and F7), the best cultivars or mixtures of phenotypically similar cultivars are tested for potential release as new cultivars.

Mass and recurrent selections can be used to improve populations of either self- or cross-pollinating crops. A genetically variable population of heterozygous individuals is either identified or created by intercrossing several different parents. The best plants are selected based on individual superiority, outstanding progeny, or excellent combining ability. The selected plants are intercrossed to produce a new population in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simply inherited, highly heritable trait into a desirable homozygous cultivar or line that is the recurrent parent. The source of the trait to be transferred is called the donor parent. After the initial cross, individuals possessing the phenotype of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent. The resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent.

The single-seed descent procedure in the strict sense refers to planting a segregating population, harvesting a sample of one seed per plant, and using the one-seed sample to plant the next generation. When the population has been advanced from the F2 to the desired level of inbreeding, the plants from which cultivars are derived will each trace to different F2 individuals. The number of plants in a population declines each generation due to failure of some seeds to germinate or some plants to produce at least one seed. As a result, not all of the F2 plants originally sampled in the population will be represented by a progeny when generation advance is completed.

Descriptions of other breeding methods that are commonly used for different traits and crops can be found in one of several reference books (e.g., “Principles of Plant Breeding” John Wiley and Son, pp. 115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr, 1987).

Proper testing should detect any major faults and establish the level of superiority or improvement over current cultivars. In addition to showing superior performance, there must be a demand for a new cultivar that is compatible with industry standards or which creates a new market. The introduction of a new cultivar will incur additional costs to the seed producer, the grower, processor and consumer for special advertising and marketing, altered seed and commercial production practices, and new product utilization. The testing preceding release of a new cultivar should take into consideration research and development costs as well as technical superiority of the final cultivar. For seed-propagated cultivars, it must be feasible to produce seed easily and economically.

Lettuce in general and romaine lettuce in particular is an important and valuable vegetable crop. Thus, a continuing goal of lettuce plant breeders is to develop stable, high yielding lettuce cultivars that are agronomically sound. To accomplish this goal, the lettuce breeder must select and develop lettuce plants with traits that result in superior cultivars.

The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

According to the invention, there is provided a novel romaine lettuce cultivar designated Green Thunder. This invention thus relates to the seeds of lettuce cultivar Green Thunder, to the plants of lettuce cultivar Green Thunder and to methods for producing a lettuce plant produced by crossing the lettuce Green Thunder with itself or another lettuce cultivar, and to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic lettuce plants produced by that method. This invention also relates to methods for producing other lettuce cultivars derived from lettuce cultivar Green Thunder and to the lettuce cultivar derived by the use of those methods. This invention further relates to hybrid lettuce seeds and plants produced by crossing the cultivar Green Thunder with another lettuce cultivar.

In another aspect, the present invention provides regenerable cells for use in tissue culture of lettuce cultivar Green Thunder. The tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing lettuce plant, and of regenerating plants having substantially the same genotype as the foregoing lettuce plant. Preferably, the regenerable cells in such tissue cultures will be embryos, protoplasts, seeds, callus, pollen, leaves, anthers, pistils, roots, root tips and meristematic cells. Still further, the present invention provides lettuce plants regenerated from the tissue cultures of the invention.

Another aspect of the invention is to provide methods for producing other lettuce plants derived from lettuce cultivar Green Thunder. Lettuce cultivars derived by the use of those methods are also part of the invention.

The invention also relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic lettuce plant produced by that method.

In another aspect, the present invention provides for single gene converted plants of Green Thunder. The single transferred gene may preferably be a dominant or recessive allele. Preferably, the single transferred gene will confer such traits as male sterility, herbicide resistance, insect resistance, resistance for bacterial, fungal, or viral disease, male fertility, enhanced nutritional quality and industrial usage. The single gene may be a naturally occurring lettuce gene or a transgene introduced through genetic engineering techniques.

The invention further provides methods for developing lettuce plants in a lettuce plant breeding program using plant breeding techniques including recurrent selection, backcrossing, pedigree breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection and transformation. Seeds, lettuce plants, and parts thereof produced by such breeding methods are also part of the invention.

In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by study of the following descriptions.

DEFINITIONS

In the description and tables which follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:

Allele. The allele is any of one or more alternative form of a gene, all of which alleles relate to one trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedly crosses hybrid progeny back to one of the parents, for example, a first generation hybrid F1 with one of the parental genotype of the F1 hybrid.

Essentially all the physiological and morphological characteristics. A plant having essentially all the physiological and morphological characteristics means a plant having essentially all of the physiological and morphological characteristics of the recurrent parent, except for the characteristics derived from the converted gene.

Regeneration. Regeneration refers to the development of a plant from tissue culture.

Single gene converted. Single gene converted or conversion plant refers to plants which are developed by a plant breeding technique called backcrossing wherein essentially all of the desired morphological and physiological characteristics of a cultivar are recovered in addition to the single gene transferred into the cultivar via the backcrossing technique or via genetic engineering.

Maturity Date. Maturity refers to the stage when the plants are of full size or optimum weight, in marketable form or shape to be of commercial or economic value. In romaine types they range from 50-75 days from time of seeding, depending upon the season of the year.

RHS. RHS refers to the Royal Horticultural Society of England which publishes an official botanical color chart quantitatively identifying colors according to a defined numbering system, The chart may be purchased from Royal Horticulture Society Enterprise Ltd RHS Garden; Wisley, Woking; Surrey GU236QB, UK.

Yield (Tons/Acre). The yield in tons/acre is the actual yield of the lettuce at harvest.

DETAILED DESCRIPTION OF THE INVENTION

Lettuce cultivar Green Thunder has superior characteristics and was developed from the cross 00BS-0719, a proprietary romaine breeding line, and Green Forest, which was made in the summer of 2000 in the greenhouse at Synergene Seed in California. The F1 hybrids were grown in a greenhouse during the fall and winter of 2000 and 2001. F2 selection was made at Synergene Seed Spring Nursery in the spring of 2001. The F3 selections were made in the spring of 2002 at Synergene Seed Spring Nursery. F4 plants were selected in 2003 in the Synergene Seed Spring Nursery. F4 plants were selected and bulked in field plots in San Joaquin Valley, Calif. during the summers of 2003 and 2004.

Green Thunder is a romaine lettuce with a very dark green leaf color, thick and slightly blistered leaf texture, highly dense and V-shaped head; it has a very vigorous growth habit and is widely adoptable in a variety of environments. Green Thunder is resistant to tipburn and Sclerotinia. It is also highly tolerant to twisting and mid-rib deformity. Green Thunder has shown a very good adaptability in the coastal California and desert Arizona regions of the United States.

Some of the criteria used for selection in various generations include: color, disease resistance, head weight, number of leaves, appearance and length, yield, emergence, maturity, plant architecture, seed yield and quality.

The cultivar has shown uniformity and stability for the traits, within the limits of environmental influence for the traits. It has been self-pollinated a sufficient number of generations with careful attention to uniformity of plant type. The cultivar has been increased with continued observation for uniformity. No variant traits have been observed or are expected in Green Thunder.

Lettuce cultivar Green Thunder has the following morphologic and other characteristics (based primarily on data collected at Salinas, Calif.).

TABLE 1

VARIETY DESCRIPTION INFORMATION

Plant Type

Romaine

Seed

Color: Black

Light dormancy: Light not required

Heat dormancy: Susceptible

Cotyledon to Fourth Leaf Stage

Shape of cotyledons: Very Broad

Undulation: Flat

Anthocyanin distribution: Absent

Rolling: Absent

Cupping: Uncupped

Reflexing: None

Mature Leaves

Margin - Incision depth: Absent/Shallow

Margin - Indentation: Entire

Margin - Undulation of the apical margin: Absent/Slightly

Green color: Very dark green

Anthocyanin - Distribution: Absent

Size: Large

Glossiness: Glossy

Blistering: Moderate

Trichomes: Absent

Leaf thickness: Thick

Plant at Market Stage

Head shape: Non-heading, V-shaped

Head size class: Large

Head weight: 940 g

Head firmness: Loose

Core

Diameter at base of head: 4.3 cm

Core height from base of head to apex: 6.7 cm

Maturity

Summer: 52 days

Winter: 92 days

Adaptation

Primary Regions of Adaptation (tested and proven adapted)

Southwest (California, Arizona desert): Adapted

West Coast: Adapted

Southeast: N/A

Northeast: Adapted

Spring area: San Joaquin, Imperial, CA; Yuma, AZ

Summer area: Salinas, Santa Maria, San Juan Bautista, CA

Fall area: Salinas, Santa Maria, Oxnard, CA

Winter area: Yuma, AZ; Imperial, Coachella, CA

Greenhouse: N/A

Soil Type: Both Mineral and Organic

Disease and Stress Reactions

Virus

Big Vein: Intermediate

Lettuce Mosaic: Susceptible

Cucumber Mosaic: Not tested

Broad Bean Wilt: Not tested

Turnip Mosaic: Not tested

Best Western Yellows: Not tested

Lettuce Infectious Yellows: Not tested

Fungal/Bacterial

Corky Root Rot (Pythium Root Rot): Intermediate

Downy Mildew: Susceptible

Powdery Mildew: Not tested

Sclerotinia Rot: Highly resistant

Bacterial Soft Rot (Pseudomonas sp. & others): Not tested

Botrytis (Gray Mold): Susceptible

Insects

Cabbage Loopers: Susceptible

Root Aphids: Susceptible

Green Peach Aphid: Susceptible

Physiological/Stress

Tipbum: Highly resistant

Heat: Intermediate

Drought: Not tested

Cold: Resistant

Salt: Not tested

Brown Rib: Resistant

Post Harvest

Pink Rib: Resistant

Russet Spotting: Not tested

Rusty Brown Discoloration: Not tested

Internal Rib Necrosis (Blackheart, Gray Rib, Gray Streak): Resistant

Brown Stain: Not tested

FURTHER EMBODIMENTS OF THE INVENTION

This invention also is directed to methods for producing a lettuce cultivar plant by crossing a first parent lettuce plant with a second parent lettuce plant wherein either the first or second parent lettuce plant is a lettuce plant of the cultivar Green Thunder. Further, both first and second parent lettuce plants can come from the cultivar Green Thunder. Still further, this invention also is directed to methods for producing a cultivar Green Thunder-derived lettuce plant by crossing cultivar Green Thunder with a second lettuce plant and growing the progeny seed, and repeating the crossing and growing steps with the cultivar Green Thunder-derived plant from 0 to 7 times. Thus, any such methods using the cultivar Green Thunder are part of this invention: selfing, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using cultivar Green Thunder as a parent are within the scope of this invention, including plants derived from cultivar Green Thunder. Advantageously, the cultivar is used in crosses with other, different, cultivars to produce first generation (F1) lettuce seeds and plants with superior characteristics.

As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which lettuce plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants, such as embryos, pollen, ovules, flowers, seeds, roots, anthers, pistils and the like.

As is well known in the art, tissue culture of lettuce can be used for the in vitro regeneration of a lettuce plant. Tissue culture of various tissues of lettuces and regeneration of plants therefrom is well known and widely published. For example, reference may be had to Teng et al., HortScience. 1992, 27: 9, 1030-1032, Teng et al., HortScience. 1993, 28: 6, 669-1671, Zhang et al., Journal of Genetics and Breeding. 1992, 46: 3, 287-290, Webb et al., Plant Cell Tissue and Organ Culture. 1994, 38: 1, 77-79, Curtis et al., Journal of Experimental Botany. 1994, 45: 279, 1441-1449, Nagata et al., Journal for the American Society for Horticultural Science. 2000, 125: 6, 669-672. It is clear from the literature that the state of the art is such that these methods of obtaining plants are “conventional” in the sense that they are routinely used and have a very high rate of success. Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce lettuce plants having the physiological and morphological characteristics of variety Green Thunder.

With the advent of molecular biological techniques that have allowed the isolation and characterization of genes that encode specific protein products, scientists in the field of plant biology developed a strong interest in engineering the genomes of plants to contain and express foreign genes, or additional, or modified versions of native, or endogenous, genes (perhaps driven by different promoters) in order to alter the traits of a plant in a specific manner. Such foreign additional and/or modified genes are referred to herein collectively as “transgenes”. Over the last fifteen to twenty years several methods for producing transgenic plants have been developed, and the present invention in particular embodiments also relates to transformed versions of the claimed cultivar.

Plant transformation involves the construction of an expression vector that will function in plant cells. Such a vector comprises DNA comprising a gene under control of, or operatively linked to, a regulatory element (for example, a promoter). The expression vector may contain one or more such operably linked gene/regulatory element combinations. The vector(s) may be in the form of a plasmid, and can be used alone or in combination with other plasmids, to provide transformed lettuce plants, using transformation methods as described below to incorporate transgenes into the genetic material of the lettuce plant(s).

Expression Vectors for Lettuce Transformation-Markers

Expression vectors include at least one genetic marker, operably linked to a regulatory element (a promoter, for example) that allows transformed cells containing the marker to be either recovered by negative selection, i.e., inhibiting growth of cells that do not contain the selectable marker gene, or by positive selection, i.e., screening for the product encoded by the genetic marker. Many commonly used selectable marker genes for plant transformation are well known in the transformation arts, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or an herbicide, or genes that encode an altered target which is insensitive to the inhibitor. A few positive selection methods are also known in the art.

Another class of marker genes for plant transformation requires screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include β-glucuronidase (GUS), α-galactosidase, luciferase and chloramphenicol acetyltransferase. Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock et al., EMBO J. 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not require destruction of plant tissue are available. Molecular Probes publication 2908, Imagene Green, p. 1-4 (1993) and Naleway et al., J. Cell Biol. 115:151a (1991). However, these in vivo methods for visualizing GUS activity have not proven useful for recovery of transformed cells because of low sensitivity, high fluorescent backgrounds and limitations associated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has been utilized as a marker for gene expression in prokaryotic and eukaryotic cells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFP may be used as screenable markers.

Expression Vectors for Lettuce Transformation-Promoters

Genes included in expression vectors must be driven by a nucleotide sequence comprising a regulatory element, for example, a promoter. Several types of promoters are well known in the transformation arts, as are other regulatory elements that can be used alone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as “tissue-preferred”. Promoters which initiate transcription only in certain tissue are referred to as “tissue-specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most environmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression in lettuce. Optionally, the inducible promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in lettuce. With an inducible promoter the rate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promoters include, but are not limited to, that from the ACEI system which responds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 gene from maize which responds to benzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genetics 227:229-237 (1991)). A particularly preferred inducible promoter is a promoter that responds to an inducing agent to which plants do not normally respond. An exemplary inducible promoter is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone. Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression in lettuce or the constitutive promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in lettuce.

A tissue-specific promoter is operably linked to a gene for expression in lettuce. Optionally, the tissue-specific promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in lettuce. Plants transformed with a gene of interest operably linked to a tissue-specific promoter produce the protein product of the transgene exclusively, or preferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in the instant invention. Exemplary tissue-specific or tissue-preferred promoters include, but are not limited to, a root-preferred promoter, such as that from the phaseolin gene (Murai et al., Science 23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324 (1985)); a leaf-specific and light-induced promoter such as that from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985) and Timko et al., Nature 318:579-582 (1985)); an anther-specific promoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics 217:240-245 (1989)); a pollen-specific promoter such as that from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or a microspore-preferred promoter such as that from apg (Twell et al., Sex. Plant Reprod. 6:217-224 (1993).

Transport of protein produced by transgenes to a subcellular compartment such as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall or mitochondrion or for secretion into the apoplast, is accomplished by means of operably linking the nucleotide sequence encoding a signal sequence to the 5′ and/or 3′ region of a gene encoding the protein of interest. Targeting sequences at the 5′ and/or 3′ end of the structural gene may determine, during protein synthesis and processing, where the encoded protein is ultimately compartmentalized.

With transgenic plants according to the present invention, a foreign protein can be produced in commercial quantities. Thus, techniques for the selection and propagation of transformed plants, which are well understood in the art, yield a plurality of transgenic plants which are harvested in a conventional manner, and a foreign protein then can be extracted from a tissue of interest or from total biomass. Protein extraction from plant biomass can be accomplished by known methods which are discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6 (1981).

According to a preferred embodiment, the transgenic plant provided for commercial production of foreign protein is lettuce. In another preferred embodiment, the biomass of interest is seed. For the relatively small number of transgenic plants that show higher levels of expression, a genetic map can be generated, primarily via conventional RFLP, PCR and SSR analysis, which identifies the approximate chromosomal location of the integrated DNA molecule. For exemplary methodologies in this regard, see Glick and Thompson, Methods in Plant Molecular Biology and Biotechnology CRC Press, Boca Raton 269:284 (1993). Map information concerning chromosomal location is useful for proprietary protection of a subject transgenic plant. If unauthorized propagation is undertaken and crosses made with other germplasm, the map of the integration region can be compared to similar maps for suspect plants, to determine if the latter have a common parentage with the subject plant. Map comparisons would involve hybridizations, RFLP, PCR, SSR and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can be expressed in transformed plants. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest. Exemplary genes implicated in this regard include, but are not limited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode

A. Plant disease resistance genes. Plant defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence (Avr) gene in the pathogen. A plant cultivar can be transformed with a cloned resistance gene(s) to engineer plants that are resistant to specific pathogen strains. See, for example Jones et al., Science 266:789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance to Pseudomonas syringae).

F. An insect-specific hormone or pheromone such as an ecdysteroid or juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof. See, for example, the disclosure by Hammock et al., Nature 344:458 (1990), of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone.

H. An insect-specific venom produced in nature by a snake, a wasp, etc. For example, see Pang et al., Gene 116:165 (1992), for disclosure of heterologous expression in plants of a gene coding for a scorpion insectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, a sesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including the post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic. See PCT application WO 93/02197 in the name of Scott et al., which discloses the nucleotide sequence of a callase gene. DNA molecules which contain chitinase-encoding sequences can be obtained, for example, from the ATCC under Accession Nos. 39637 and 67152. See also Kramer et al., Insect Biochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequence of a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al., Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene.

M. A membrane permease, a channel former or a channel blocker. For example, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), of heterologous expression of a cecropin-α, lytic peptide analog to render transgenic tobacco plants resistant to Pseudomonas solanacearum.

Q. A developmental-arrestive protein produced in nature by a pathogen or a parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology 10:1436 (1992). The cloning and characterization of a gene which encodes a lettuce endopolygalacturonase-inhibiting protein is described by Toubart et al., Plant J. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. For example, Logemann et al., Bio/Technology 10:305 (1992), have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease.

A. An herbicide that inhibits the growing point or meristem, such as an imidazalinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzymes as described, for example, by Lee et al., EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449 (1990), respectively.

B. Glyphosate (resistance impaired by mutant 5-enolpyruvl-3-phosphoshikimate synthase (EPSP) and aroA genes, respectively) and other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus PAT, bar, genes), and pyridinoxy or phenoxy propionic acids and cyclohexones (ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses the nucleotide sequence of a form of EPSP which can confer glyphosate resistance. A DNA molecule encoding a mutant aroA gene can be obtained under ATCC accession number 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. See also Umaballava-Mobapathie in Transgenic Research. 1999, 8:1, 33-44, that discloses Lactuca sativa resistant to glufosinate. European patent application No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in European application No. 0 242 246 to Leemans et al.; DeGreef et al., Bio/Technology 7:61 (1989), describe the production of transgenic plants that express chimeric bar genes coding for phosphinothricin acetyl transferase activity. Examples of genes conferring resistance to phenoxy propionic acids and cyclohexones, such as sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes are described by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al., Plant Cell 3:169 (1991), describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genes are available under ATCC Accession Nos. 53435, 67441, and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Increased iron content of the lettuce, for example by transforming a plant with a soybean ferritin gene as described in Goto et al., Acta Horticulturae. 2000, 521, 101-109. Parallel to the improved iron content, enhanced growth of transgenic lettuces was also observed in early development stages.

B. Decreased nitrate content of leaves, for example by transforming a lettuce with a gene coding for a nitrate reductase. See for example Curtis et al., Plant Cell Report. 1999, 18: 11, 889-896.

C. Increased sweetness of the lettuce by transferring a gene coding for monellin that elicits a flavor 100,000 times sweeter than sugar on a molar basis. See Penarrubia et al., Biotechnology. 1992, 10: 5, 561-564.

Despite the fact the host range for Agrobacterium-mediated transformation is broad, some major cereal or vegetable crop species and gymnosperms have generally been recalcitrant to this mode of gene transfer, even though some success has recently been achieved in rice and corn. Hiei et al., The Plant Journal 6:271-282 (1994) and U.S. Pat. No. 5,591,616 issued Jan. 7, 1997. Several methods of plant transformation, collectively referred to as direct gene transfer, have been developed as an alternative to Agrobacterium-mediated transformation.

Following transformation of lettuce target tissues, expression of the above-described selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants, using regeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used for producing a transgenic cultivar. The transgenic cultivar could then be crossed with another (non-transformed or transformed) cultivar in order to produce a new transgenic lettuce cultivar. Alternatively, a genetic trait which has been engineered into a particular lettuce cultivar using the foregoing transformation techniques could be moved into another cultivar using traditional backcrossing techniques that are well known in the plant breeding arts. For example, a backcrossing approach could be used to move an engineered trait from a public, non-elite inbred cultivar into an elite inbred cultivar, or from an inbred cultivar containing a foreign gene in its genome into an inbred cultivar or cultivars which do not contain that gene. As used herein, “crossing” can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.

When the terms lettuce plant, cultivar or lettuce cultivar are used in the context of the present invention, this also includes any single gene conversions of that culitvar. The term single gene converted plant as used herein refers to those lettuce plants which are developed by a plant breeding technique called backcrossing wherein essentially all of the desired morphological and physiological characteristics of a cultivar are recovered in addition to the single gene transferred into the cultivar via the backcrossing technique. Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the cultivar. The term backcrossing as used herein refers to the repeated crossing of a hybrid progeny back to one of the parental lettuce plants for that cultivar. The parental lettuce plant which contributes the gene for the desired characteristic is termed the nonrecurrent or donor parent. This terminology refers to the fact that the nonrecurrent parent is used one time in the backcross protocol and therefore does not recur. The parental lettuce plant to which the gene or genes from the nonrecurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol (Poehlman & Sleper, 1994; Fehr, 1987). In a typical backcross protocol, the original cultivar of interest (recurrent parent) is crossed to a second cultivar (nonrecurrent parent) that carries the single gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a lettuce plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the single transferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for a successful backcrossing procedure. The goal of a backcross protocol is to alter or substitute a single trait or characteristic in the original cultivar. To accomplish this, a single gene of the recurrent cultivar is modified or substituted with the desired gene from the nonrecurrent parent, while retaining essentially all of the rest of the desired genetic, and therefore the desired physiological and morphological, constitution of the original cultivar. The choice of the particular nonrecurrent parent will depend on the purpose of the backcross; one of the major purposes is to add some commercially desirable and/or agronomically important trait to the plant. The exact backcrossing protocol will depend on the characteristic or trait being altered to determine an appropriate testing protocol. Although backcrossing methods are simplified when the characteristic being transferred is a dominant allele, a recessive allele may also be transferred. In this instance it may be necessary to introduce a test of the progeny to determine if the desired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularly selected for in the development of a new cultivar but that can be improved by backcrossing techniques. Single gene traits may or may not be transgenic, examples of these traits include but are not limited to, herbicide resistance, resistance for bacterial, fungal, or viral disease, insect resistance, enhanced nutritional quality, industrial usage, yield stability and yield enhancement. These genes are generally inherited through the nucleus. Several of these single gene traits are described in U.S. Pat. Nos. 5,777,196, 5,948,957 and 5,969,212, the disclosures of which are specifically hereby incorporated by reference.

TABLES

In the tables that follow, the traits and characteristics of lettuce cultivar Green Thunder are given compared to two commercial romaine lettuce cultivars, Green Forest and PIC Cos.

Table 2 below shows the mature seed stalk height for Green Thunder as compared to the seed stalk height for Green Forest and PIC Cos. Seed stalk height is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 2, Green Thunder has a significantly taller mature seed stalk height than either Green Forest or PIC Cos.

TABLE 2

Seed Stalk Height (cm)

Green Thunder

Green Forest

PIC Cos

105

101

100

107

105

102

109

102

100

108

100

95

100

98

96

102

105

105

103

103

97

111

99

98

110

96

99

101

97

98

102

100

99

100

106

100

110

95

96

Anova: Single Factor

SUMMARY

Groups

Count

Sum

Average

Variance

Green Thunder

13

1368

105.2307692

16.85897

Green Forest

13

1307

100.5384615

12.60256

PIC Cos

13

1285

98.84615385

7.307692

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups

284.4615385

2

142.2307692

11.6046

0.000129

3.259444

Within Groups

441.2307692

36

12.25641026

Total

725.6923077

38

Table 3 below shows the seed stalk spread for Green Thunder as compared to the seed stalk spread for Green Forest and PIC Cos. Seed stalk spread is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 3, Green Thunder has a significantly wider mature seed stalk spread than Green Forest but a narrower mature seed stalk spread than PIC Cos.

TABLE 3

Seed Stalk Spread (cm)

Green Thunder

Green Forest

PIC Cos

37

44

52

50

42

45

44

42

47

45

40

41

45

40

48

46

45

41

38

44

42

42

42

47

43

35

43

46

44

48

40

36

44

45

44

45

44

40

40

Anova: Single Factor

SUMMARY

Groups

Count

Sum

Average

Variance

Green Thunder

13

565

43.46153846

12.4359

Green Forest

13

538

41.38461538

9.75641

PIC Cos

13

583

44.84615385

12.14103

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups

78.92307692

2

39.46153846

3.448096

0.042649

3.259444

Within Groups

412

36

11.44444444

Total

490.9230769

38

Tables 4 through 8 show data collected in eight different locations. Location 1 was near Bard, Calif., location 2 was near San Juan Bautista, Calif., locations 3, 5, 6, 7, and 8 were near Salinas, Calif. and location 4 was near Yuma, Ariz.

Table 4 below shows the plant weight in grams at harvest maturity of Green Thunder as compared to Green Forest and PIC Cos. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 4, Green Thunder is significantly heavier in plant weight at harvest maturity than either Green Forest or PIC Cos.

TABLE 4

Trial

Plant Weight (g) at Harvest Maturity

Location

Loc1

Loc2

Loc3

Loc4

Loc5

Loc6

Loc7

Loc8

Green

1020

908

1021

1201

1021

1201

540

805

Thunder

1160

1160

984

1136

984

1136

790

780

936

1160

908

1165

908

1165

810

810

908

1020

1025

1146

1025

1146

790

650

908

681

984

990

984

990

810

820

846

1076

1026

1025

1026

1025

570

870

846

790

988

914

988

914

85

650

1020

965

956

986

956

986

860

750

1244

681

1024

1034

1024

1034

820

750

1104

795

1136

1130

1136

1130

790

650

Green

820

622

756

759

756

759

670

680

Forest

795

1076

687

774

687

774

710

690

1076

681

909

804

909

804

610

720

874

795

675

907

675

907

580

820

908

795

756

682

756

682

690

750

820

1076

764

673

764

673

620

800

908

622

904

698

904

698

710

720

1244

795

689

768

6889

768

550

780

681

681

677

759

677

759

690

820

846

846

757

683

757

683

710

810

PIC Cos

846

760

688

766

688

766

610

650

965

820

901

771

901

771

710

720

875

795

679

699

679

699

540

590

705

622

766

706

766

706

600

680

846

740

752

755

752

755

580

710

965

622

742

899

742

899

570

800

875

908

911

878

911

878

810

800

622

908

903

756

903

756

580

720

622

681

699

766

699

766

720

740

705

622

698

751

698

751

710

580

Anova: Two-Factor With Replication

SUM-

MARY

Loc1

Loc2

Loc3

Loc4

Loc5

Loc6

Loc7

Loc8

Total

Green

Thunder

Count

10

10

10

10

10

10

10

10

80

Sum

9992

9236

10052

10727

10052

10727

6865

7535

75186

Average

999.2

923.6

1005.2

1072.7

1005.2

1072.7

686.5

753.5

939.825

Variance

18355.73

33149.16

3511.067

8995.344444

3511.067

8995.344

56378.06

6300.278

34406.96

Green

Forest

Count

10

10

10

10

10

10

10

10

80

Sum

8972

7989

7574

7507

13774

7507

6540

7590

67453

Average

897.2

798.9

757.4

750.7

1377.4

750.7

654

759

843.1625

Variance

24971.07

27357.88

7490.044

5152.011111

3757250

5152.011

3515.556

2921.111

482030

PIC Cos

Count

10

10

10

10

10

10

10

10

80

Sum

8026

7478

7739

7747

7739

7747

6430

6990

59896

Average

802.6

747.8

773.9

774.7

773.9

774.7

643

699

748.7

Variance

16809.16

12324.18

8992.544

4226.233333

8992.544

4226.233

7734.444

5765.556

10295.5

Total

Count

30

30

30

30

30

30

30

30

Sum

26990

24703

25365

25981

31565

25981

19835

22115

Average

899.6667

823.4333

845.5

866.0333333

1052.167

866.0333

661.1667

737.1667

Variance

25330.09

28242.67

19443.64

27893.41264

1233860

27893.41

21340.83

5409.799

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Sample

1461215

2

730607.6

4.338013203

0.014219

3.03767

Columns

2780088

7

397155.5

2.358127411

0.024316

2.052154

Interaction

2453089

14

175220.6

1.040379891

0.414431

1.737799

Within

36378690

216

168419.9

Total

43073082

239

Table 5 below shows the leaf width of Green Thunder at harvest maturity as compared to Green Forest and PIC Cos. Leaf width is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 5, Green Thunder is significantly wider in leaf width at harvest maturity than Green Forest but not significantly wider than PIC Cos.

TABLE 5

Leaf Width (cm) at Harvest Maturity

Location:

Loc1

Loc2

Loc3

Loc4

Loc5

Loc6

Loc7

Loc8

Green Thunder

20

21.5

21

18

21

18

22

20

24

21

19

21

19

21

20

22

23

21

21

18

18

18

20

20

21

21

18

18

18

18

20

19

19

16

21

20

21

20

22

19

21.5

20

18

19

18

19

20

20

16

19

18

18

18

18

22

22

21

20

20

21

20

21

20

22

23

20

18

19

17.5

19

22

22

21

19

17

20

17

18

20

20

Green Forest

20.0

16.0

15.0

16.0

15.0

16.0

19.5

22.0

20

20

14

17

14

17

19.5

19

20

16

13

13

17

13

20

20

18

19

17

16

17

16

20

21

19

15

16

13

16

13

19

21

18

21.5

13

14

13

14

19

19

19

14

16

12

16

12

20

22

20.5

16

12

14

12

14

19

22

16

17

14

14

13.5

14

19

19

21

18

13.5

15

14

15

19.5

20

PIC Cos

17

20

20

20

20

20

21

22

19

19

20

16

19.5

16

20

22

17

18

19

17

17

17

21

21

15

17

17

19

17

19

22

22

19

19

20

15

20

15

20

21

17

16

16

17

16

17

22

19

16

18

15

17

15

20

20

19

14

21

17

20

17

20

20

19

17

17

17

17

17

17

21

20

17

15

17

17

17

17

21

21

Anova: Two-Factor With Replication

SUMMARY

Loc1

Loc2

Loc3

loc4

Loc5

Loc6

Loc7

Loc8

Total

Green Thunder

Count

10

10

10

10

10

10

10

10

80

Sum

209.5

198.5

191

192

187.5

190

208

206

1582.5

Average

20.95

19.85

19.1

19.2

18.75

19

20.8

20.6

19.78125

Variance

5.247222

2.558333

2.322222

1.511111111

2.069444

1.555556

1.066667

1.6

2.745847

Green Forest

Count

10

10

10

10

10

10

10

10

80

Sum

191.5

172.5

143.5

144

147.5

144

194.5

205

1342.5

Average

19.15

17.25

14.35

14.4

14.75

14.4

19.45

20.5

16.78125

Variance

2.225

5.513889

2.558333

2.488888889

2.958333

2.488889

0.191667

1.611111

8.378758

PIC Cos

Count

10

10

10

10

10

10

10

10

80

Sum

168

180

178

175

175.5

178

208

206

1468.5

Average

16.8

18

17.8

17.5

17.55

17.8

20.8

20.6

18.35625

Variance

2.4

3.333333

3.288889

2.722222222

2.913889

3.288889

0.622222

1.6

4.267049

Total

Count

30

30

30

30

30

30

30

30

Sum

569

551

512.5

511

510.5

512

610.5

617

Average

18.96667

18.36667

17.08333

17.03333333

17.01667

17.06667

20.35

20.56667

Variance

6.050575

4.774713

6.691092

6.171264368

5.370402

6.202299

1.002586

1.495402

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Sample

360.3

2

180.15

74.37029958

2.67E−25

3.03767

Columns

481.249

7

68.74985

28.38160993

1.59E−27

2.052154

Interaction

211.4667

14

15.10476

6.235612923

2.1E−10

1.737799

Within

523.225

216

2.422338

Total

1576.241

239

Table 6 below shows the leaf index of Green Thunder at harvest maturity as compared to Green Forest and PIC Cos. Leaf index is calculated by dividing the leaf length by the leaf width. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 6, Green Thunder has a significantly different leaf index than Green Forest indicating that Green Thunder has a different leaf shape than Green Forest at harvest maturity. However, Green Thunder has a similar leaf index to PIC Cos indicating that Green Thunder has a similar leaf shape at harvest maturity to that of PIC Cos.

TABLE 6

Leaf Index at Harvest Maturity

Location:

Loc1

Loc2

Loc3

Loc4

Loc5

Loc6

Loc7

Loc8

Green Thunder

1.35

1.33

1.33

1.39

1.33

1.4

1.32

1.5

1.29

1.33

1.37

1.33

1.37

1.3

1.5

1.45

1.3

1.33

1.33

1.39

1.39

1.4

1.45

1.55

1.33

1.33

1.39

1.39

1.39

1.4

1.5

1.58

1.37

1.44

1.33

1.35

1.33

1.4

1.36

1.58

1.33

1.35

1.39

1.37

1.39

1.4

1.55

1.55

1.44

1.37

1.39

1.39

1.39

1.4

1.36

1.36

1.33

1.35

1.35

1.33

1.35

1.3

1.55

1.36

1.3

1.35

1.39

1.37

1.43

1.4

1.45

1.41

1.33

1.37

1.41

1.35

1.42

1.5

1.6

1.6

Green Forest

1.35

1.44

1.47

1.44

1.47

1.4

1.49

1.45

1.35

1.35

1.5

1.41

1.5

1.4

1.54

1.58

1.35

1.44

1.54

1.54

1.42

1.5

1.5

1.55

1.39

1.37

1.53

1.44

1.41

1.4

1.5

1.43

1.21

1.47

1.44

1.54

1.44

1.5

1.53

1.48

1.39

1.33

1.54

1.5

1.54

1.5

1.58

1.58

1.21

1.5

1.44

1.58

1.44

1.6

1.5

1.45

1.34

1.44

1.58

1.5

1.58

1.5

1.58

1.36

1.44

1.41

1.5

1.5

1.56

1.5

1.58

1.58

1.33

1.39

1.56

1.47

1.5

1.5

1.59

1.5

PIC Cos

1.41

1.35

1.35

1.35

1.35

1.4

1.38

1.41

1.37

1.37

1.35

1.44

1.36

1.4

1.4

1.36

1.41

1.39

1.37

1.41

1.41

1.4

1.38

1.43

1.53

1.41

1.41

1.37

1.41

1.4

1.36

1.45

1.37

1.37

1.35

1.47

1.35

1.5

1.55

1.43

1.41

1.44

1.44

1.41

1.44

1.4

1.36

1.58

1.44

1.39

1.47

1.41

1.47

1.4

1.45

1.58

1.5

1.33

1.41

1.35

1.41

1.4

1.5

1.58

1.41

1.41

1.41

1.41

1.41

1.4

1.43

1.5

1.41

1.47

1.41

1.41

1.41

1.4

1.48

1.48

Anova: Two-Factor With Replication

SUMMARY

Loc1

Loc2

Loc3

loc4

Loc5

Loc6

Loc7

Loc8

Total

Green Thunder

Count

10

10

10

10

10

10

10

10

80

Sum

13.37

13.55

13.68

13.66

13.79

13.9

14.64

14.94

111.53

Average

1.337

1.355

1.368

1.366

1.379

1.39

1.464

1.494

1.394125

Variance

0.00189

0.001139

0.000929

0.000604444

0.001166

0.003222

0.008738

0.008582

0.005698

Green Forest

Count

10

10

10

10

10

10

10

10

80

Sum

13.36

14.14

15.1

14.92

14.86

14.8

15.39

14.96

117.53

Average

1.336

1.414

1.51

1.492

1.486

1.48

1.539

1.496

1.469125

Variance

0.005449

0.002916

0.002356

0.002795556

0.003582

0.004

0.001632

0.005716

0.006907

PIC Cos

Count

10

10

10

10

10

10

10

10

80

Sum

14.26

13.93

13.97

14.03

14.02

14.1

14.29

14.8

113.4

Average

1.426

1.393

1.397

1.403

1.402

1.41

1.429

1.48

1.4175

Variance

0.002671

0.001734

0.00169

0.001423333

0.001507

0.001

0.00421

0.006178

0.003039

Total

Count

30

30

30

30

30

30

30

30

Sum

40.99

41.62

42.75

42.61

42.67

42.8

44.32

44.7

Average

1.366333

1.387333

1.425

1.420333333

1.422333

1.426667

1.477333

1.49

Variance

0.004948

0.002413

0.005426

0.00438954

0.004129

0.004092

0.006703

0.006407

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Sample

0.235641

2

0.11782

37.63785199

9.47E−15

3.03767

Columns

0.354798

7

0.050685

16.19152694

4.53E−17

2.052154

Interaction

0.204919

14

0.014637

4.675831164

1.96E−07

1.737799

Within

0.67616

216

0.00313

Total

1.471518

239

Table 7 below shows the leaf area of Green Thunder at harvest maturity as compared to Green Forest and PIC Cos. Leaf area is calculated by multiplying the leaf length by the leaf width. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 7, Green Thunder has a significantly larger leaf area than both Green Forest and PIC Cos at harvest maturity.

TABLE 7

Leaf Area (cm2) at Harvest Maturity

Location:

Loc1

Loc2

Loc3

loc4

Loc5

Loc6

Loc7

Loc8

Green Thunder

540

613

588

450

588

450

638

600

744

588

484

588

494

588

600

704

690

588

588

450

450

450

580

620

588

588

450

450

450

450

600

570

494

368

588

540

588

540

660

570

613

540

450

494

450

494

620

620

368

494

450

450

450

450

660

660

588

540

540

588

540

588

620

660

690

540

450

494

438

494

704

682

588

494

408

540

408

486

640

640

Green Forest

540

368

330

368

330

368

566

704

540

540

294

408

294

408

585

570

540

368

260

260

408

260

600

620

450

494

442

368

408

368

600

630

437

330

368

260

368

260

551

651

450

613

260

294

260

294

570

570

437

294

368

228

368

228

600

704

564

368

228

294

228

294

570

660

368

408

294

294

284

294

570

570

588

450

284

330

294

330

605

600

PIC Cos

408

540

540

540

540

540

609

682

494

494

540

368

517

368

560

660

408

450

494

408

408

408

609

630

345

408

408

494

408

494

660

704

494

494

540

330

330

330

620

630

408

368

368

408

368

408

660

570

368

450

330

408

330

540

580

570

294

588

408

540

408

540

600

570

408

408

108

108

108

108

630

600

408

330

408

108

108

108

651

651

Anova: Two-Factor With Replication

SUMMARY

Loc1

Loc2

Loc3

loc4

Loc5

Loc6

Loc7

Loc8

Total

Green Thunder

Count

10

10

10

10

10

10

10

10

80

Sum

5903

5353

4996

5044

4856

4990

6322

6326

43790

Average

590.3

535.3

499.6

504.4

485.6

499

632.2

632.6

547.375

Variance

11695.12

5092.9

4838.933

3174.044444

4150.933

3038.444

1321.289

2034.711

7410.668

Green Forest

Count

10

10

10

10

10

10

10

10

80

Sum

4914

4233

3128

3104

3242

3104

5817

6279

33821

Average

491.4

423.3

312.8

310.4

324.2

310.4

581.7

627.9

422.7625

Variance

5140.267

10016.46

4153.956

3246.933333

3859.067

3246.933

350.9

2672.1

18801.12

PIC Cos

Count

10

10

10

10

10

10

10

10

80

Sum

4035

4530

4144

3712

3525

3844

6179

6267

36236

Average

403.5

453

414.4

371.2

352.5

384.4

617.9

626.7

452.95

Variance

3683.833

6175.333

17438.04

24042.84444

21363.39

26864.71

1117.656

2345.789

22218.96

Total

Count

30

30

30

30

30

30

30

30

Sum

14852

14116

12268

11860

11623

11938

18318

18872

Average

495.0667

470.5333

408.9333

395.3333333

387.4333

397.9333

610.6

629.0667

Variance

12391.24

8927.361

14234.41

16244.50575

14238.46

16515.44

1333.076

2195.444

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Sample

676150.4

2

338075.2

47.43123725

8.39E−18

3.03767

Columns

2005861

7

286551.6

40.20258531

6.98E−36

2.052154

Interaction

280586.4

14

20041.89

2.811834592

0.000701

1.737799

Within

1539581

216

7127.691

Total

4502179

239

Table 8 below shows the core length of Green Thunder at harvest maturity as compared to Green Forest and PIC Cos. Core length is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 8, Green Thunder has a significantly longer core than both Green Forest and PIC Cos at harvest maturity.

TABLE 8

Core Length (cm) at Harvest Maturity

Location:

Loc1

Loc2

Loc3

Loc4

Loc5

Loc6

Loc7

Loc8

Green Thunder

7.7

9.2

5

6.5

5

6.5

5.5

7

8.6

9

7

7

7

7

6

6.5

8.4

10

7

6

7

6

6.5

6.5

9

9.5

7

6

7

6

6.5

4

8.2

5.5

6.5

7

6.5

7

7

4

8

7.5

6

7

6

7

6

6.5

7

7

6

5.5

6

5.5

5.8

6.8

7.6

8

5.5

5

5.5

5

6.2

7

9

6

6

6

6

6

7

7

8

6.5

6.5

6.5

6.5

6.5

6.5

6.5

Green Forest

7

5.5

5

5

5

5

6.5

6.2

7

9

5

5

5

5

7

7

8.2

7.8

5

5

5

5

6

6.5

7.7

10

5

6

5

6

6.5

6.5

9

6.5

5

5

5

5

6

6.2

7

8

5

5

5

5

6.5

6

9.5

6

5

5

5

5

6.5

6.5

10

7

5

5

5

5

6.5

7

5

6

5

5

5

5

7

7

8.5

9

6

5

6

5

7

6.5

PIC Cos

7

6

5.5

6

5.5

6

5

6.5

7.5

6

6

5

6

5

5

4.5

6.5

6

5.5

5

5.5

5

4

4.2

5

5

5

6

5

6

5

5.6

7

6

6

5

6

5

4

5

6

7

5

6

5

6

4

5

6

7

5

5

5

5

5

5

4

8

4

6

4

5

5

4

5.5

5

5

5

5

5

4

4

5.5

5

5

5

4

4

5

4

Anova: Two-Factor With Replication

SUMMARY

Loc1

Loc2

Loc3

loc4

Loc5

Loc6

Loc7

Loc8

Total

Green Thunder

Count

10

10

10

10

10

10

10

10

80

Sum

81.5

78.2

62.5

62.5

62.5

62.5

63

61.8

534.5

Average

8.15

7.82

6.25

6.25

6.25

6.25

6.3

6.18

6.68125

Variance

0.398333

2.457333

0.458333

0.458333333

0.458333

0.458333

0.242222

1.368444

1.299264

Green Forest

Count

10

10

10

10

10

10

10

10

80

Sum

78.9

74.8

51

51

51

51

65.5

65.4

488.6

Average

7.89

7.48

5.1

5.1

5.1

5.1

6.55

6.54

6.1075

Variance

2.167667

2.315111

0.1

0.1

0.1

0.1

0.136111

0.129333

1.789563

PIC Cos

Count

10

10

10

10

10

10

10

10

80

Sum

60

61

52

54

51

52

46

47.8

423.8

Average

6

6.1

5.2

5.4

5.1

5.2

4.6

4.78

5.2975

Variance

1.111111

0.988889

0.344444

0.266666667

0.488889

0.4

0.266667

0.668444

0.764804

Total

Count

30

30

30

30

30

30

30

30

Sum

220.4

214

165.5

167.5

164.5

165.5

174.5

175

Average

7.346667

7.133333

5.516667

5.583333333

5.483333

5.516667

5.816667

5.8333333

Variance

2.090851

2.36023

0.560057

0.501436782

0.629023

0.577299

0.976609

1.2685057

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Sample

77.33475

2

38.66737

58.06275418

6.61E−21

3.03767

Columns

121.8153

7

17.40218

26.13104102

9.34E−26

2.052154

Interaction

38.77458

14

2.769613

4.158838409

1.94E−06

1.737799

Within

143.847

216

0.665958

Total

381.7716

239

Table 9 below shows the length of the fourth true leaf of Green Thunder as compared to Green Forest and PIC Cos. Leaf length is measured in centimeters of a 20-day old seedling. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 9, Green Thunder has a significantly shorter fourth leaf length than Green Forest but about the same fourth leaf length as PIC Cos.

TABLE 9

4th Leaf Length (cm)

Green Thunder

Green Forest

PIC Cos

3.8

5.6

3.8

4.3

5.5

3.9

3.9

4.8

3.6

4.1

5.8

4.2

3.9

4

3.7

4.4

4.8

3.8

4.6

5.5

3.9

3.5

5.5

4

4.1

5.6

4.1

4.3

5.8

4

3.8

5.9

3.9

4

4.5

3.9

4.1

4.6

4

4.2

5.4

4.1

4

5.5

4.5

Anova: Single Factor

SUMMARY

Groups

Count

Sum

Average

Variance

Green Thunder

15

61

4.066666667

0.075238

Green Forest

15

78.8

5.253333333

0.321238

PIC Cos

15

59.4

3.96

0.046857

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups

15.46133333

2

7.730666667

52.31278

3.96E−12

3.219938

Within Groups

6.206666667

42

0.147777778

Total

21.668

44

Table 10 below shows the width of the fourth true leaf of Green Thunder as compared to Green Forest and PIC Cos. Leaf width is measured in centimeters of a 20-day old seedling. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 10, Green Thunder has a significantly narrower fourth leaf width than Green Forest but about the same fourth leaf width as PIC Cos.

TABLE 10

4th Leaf Width (cm)

Green Thunder

Green Forest

PIC Cos

2.6

2.8

2.3

2.8

3.2

2.5

2.5

2.7

2.2

2.6

3

2.5

2.4

2.1

2.1

2.5

2.6

2.4

2.6

2.8

2.3

2.2

2.9

2.5

2.6

2.9

2.5

2.8

3

2.4

2.6

3

2.4

2.5

2.4

2.2

2.5

2.5

2.4

2.7

3

2.5

2.8

3

2.6

Anova: Single Factor

SUMMARY

Groups

Count

Sum

Average

Variance

Green Thunder

15

38.7

2.58

0.026

Green Forest

15

41.9

2.793333333

0.083524

PIC Cos

15

35.8

2.386666667

0.01981

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups

1.241333333

2

0.620666667

14.39691

1.73E−05

3.219938

Within Groups

1.810666667

42

0.043111111

Total

3.052

44

Table 11 below shows the leaf index of the fourth true leaf of Green Thunder as compared to Green Forest and PIC Cos. Leaf index is calculated by dividing the leaf length by the leaf width of the fourth true leaf of a 20-day old seedling. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 11, Green Thunder has a significantly different leaf index than both Green Forest and PIC Cos indicating that Green Thunder has a different fourth true leaf shape than either Green Forest or PIC Cos.

TABLE 11

4th Leaf Index

Green Thunder

Green Forest

PIC Cos

1.46

2.07

1.65

1.54

1.72

1.56

1.56

1.78

1.64

1.58

1.93

1.68

1.63

1.9

1.76

1.76

1.85

1.58

1.77

1.96

1.7

1.59

1.9

1.6

1.58

1.93

1.64

1.54

1.93

1.67

1.46

1.97

1.63

1.6

1.88

1.73

1.71

1.84

1.67

1.56

1.8

1.64

1.43

1.83

1.73

Anova: Single Factor

SUMMARY

Groups

Count

Sum

Average

Variance

Green Thunder

15

23.77

1.584666667

0.010241

Green Forest

15

28.29

1.886

0.007526

PIC Cos

15

24.88

1.658666667

0.003155

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups

0.739791111

2

0.369895556

53.03947

3.22E−12

3.219938

Within Groups

0.292906667

42

0.006973968

Total

1.032697778

44

Table 12 below shows the cotyledon length of Green Thunder as compared to Green Forest and PIC Cos. Cotyledon length was measured in millimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 12, Green Thunder has a significantly shorter cotyledon length than Green Forest but similar cotyledon length to PIC Cos.

TABLE 12

Cotyledon length (mm)

Green Thunder

Green Forest

PIC Cos

15

19

14

15

20

14

15

19

14

16

19

12

16

20

14

15

18

14

13

18

14

13

19

16

15

18

16

15

19

16

15

19

14

15

19

14

Anova: Single Factor

SUMMARY

Groups

Count

Sum

Average

Variance

Green Thunder

12

178

14.83333

0.878788

Green Forest

12

227

18.91667

0.44697

PIC Cos

12

172

14.33333

1.333333

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups

151.7222222

2

75.86111

85.58689

8.72E−14

3.284924

Within Groups

29.25

33

0.886364

Total

180.9722222

35

Table 13 below shows the cotyledon width of Green Thunder as compared to Green Forest and PIC Cos. Cotyledon width was measured in millimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 13, Green Thunder has a significantly wider cotyledon width than both Green Forest and PIC Cos.

TABLE 13

Cotyledon Width (mm)

Green Thunder

Green Forest

PIC Cos

10

9

9

10

9

8

10

9

9

10

9

8

10

9

8

11

9

8

9

9

8

9

9

8

10

9

9

10

9

9

10

9

8

10

9

8

Anova: Single Factor

SUMMARY

Groups

Count

Sum

Average

Variance

Green Thunder

12

119

9.916667

0.265152

Green Forest

12

108

9

0

PIC Cos

12

100

8.333333

0.242424

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups

15.16666667

2

7.583333

44.8209

3.92E−10

3.284924

Within Groups

5.583333333

33

0.169192

Total

20.75

35

Table 14 below shows the cotyledon index of Green Thunder as compared to Green Forest and PIC Cos. Cotyledon index is calculated by dividing the cotyledon leaf length by the cotyledon leaf width. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 14, Green Thunder has a significantly different cotyledon index than both Green Forest and PIC Cos indicating that Green Thunder has a different cotyledon leaf shape than both Green Forest and PIC Cos.

TABLE 14

Cotyledon Width (mm)

Green Thunder

Green Forest

PIC Cos

1.5

2.11

1.56

1.5

2.22

1.75

1.5

2.11

1.56

1.6

2.11

1.5

1.6

2.22

1.75

1.36

2

1.75

1.44

2

1.75

1.44

2.11

1.75

1.5

2

1.78

1.5

2.11

1.78

1.5

2.11

1.75

1.5

2.11

1.75

Anova: Single Factor

SUMMARY

Groups

Count

Sum

Average

Variance

Green Thunder

12

17.94

1.495

0.004227

Green Forest

12

25.21

2.100833

0.005408

PIC Cos

12

20.43

1.7025

0.009948

ANOVA

Source of Variation

SS

df

MS

F

P-value

F crit

Between Groups

2.275038889

2

1.137519

174.2583

2.89E−18

3.284924

Within Groups

0.215416667

33

0.006528

Total

2.490455556

35

DEPOSIT INFORMATION

A deposit of the Synergene Seed & Technology, Inc. Proprietary lettuce cultivar designated Green Thunder disclosed above and recited in the appended claims has been made with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas. Va. 20110. The date of deposit was Aug. 14, 2007. The deposit of 2,500 seeds was taken from the same deposit maintained by Synergene Seed & Technology, Inc. since prior to the filing date of this application. All restrictions upon the deposit have been removed, and the deposit is intended to meet all of the requirements of 37 C.F.R. 1.801-1.809. The ATCC accession number is PTA-8605. The deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced as necessary during that period.

While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.